The primary developmental events of plants originate from the shoot apical meristem (SAM) (Clark, “Organ Formation at the Vegetative Shoot Meristem,” Plant Cell 9:1067-1076 (1997); Kerstetter et al., “Shoot Meristem Formation in Vegetative Development,” Plant Cell 9:1001-1010 (1997)). The shoot apical meristem (SAM) is responsible for the formation of vegetative organs such as leaves, and may undergo a phase change to form the inflorescence or floral meristem. Many of these events are controlled at the molecular level by transcription factors. Transcription factors (TFs) are proteins that act as developmental switches by binding to the DNA (or to other proteins that bind to the DNA) of specific target genes to modulate their expression. An important family of TFs involved in regulating the developmental events in apical meristems is the knox (knotted-like homeobo) gene family (Reiser et al., “Knots in the Family Tree Evolutionary Relationships and Functions of Knox Homeobox Genes,” Plant Mol Biol 42:151-166 (2000)). Knox genes have been isolated from several plant species (reviewed in Reiser et al., “Knots in the Family Tree: Evolutionary Relationships and Functions of knox Homeobox Genes,” Plant Mol. Biol. 42:151-166 (2000)) and can be divided into two classes based on expression patterns and sequence similarity (Kerstetter et al., “Sequence Analysis and Expression Patters Divide the Maize knotted1-like Homeobox Genes into Two Classes,” Plant Cell 6:1888-1887 (1994)). Class I knox genes have high similarity to the kn1 homeodomain and generally have a meristem-specific mRNA expression pattern. Class II knox genes usually have a more widespread expression pattern.
Knox genes belong to the group of TFs known as the TALE superclass (Bürglin, “Analysis of TALE Superclass Homeobox Genes (MEIS, PBC, KNOX, Iroquois, TGIF) Reveals a Novel Domain Conserved Between Plants and Animals,” Nucleic Acids Res 25:4173-4180 (1997)). These TFs are distinguished by a very high level of sequence conservation in the DNA-binding region, designated the homeodomain, and consisting of three α-helices similar to the bacterial helix-loop-helix motif (Kerstetter et al., “Sequence Analysis and Expression Patterns Divide the Maize knotted1-like Homeobox Genes into Two Classes,” Plant Cell 6:1877-1887 (1994)). The third helix, the recognition helix, is involved in DNA-binding (Mann et al., “Extra Specificity From extradenticle: the Partnership Between HOX and PBX/EXD Homeodomain Proteins,” Trends in Genet. 12:258-262 (1996)). TALE TFs contain a three amino acid loop extension (TALE), proline-tyrosine-proline, between helices I and II in the homeodomain, that has been implicated in protein interactions (Passner et al., “Structure of DNA-Bound Ultrabithorax-Extradenticle Homeodomain Complex,” Nature 397:714-719 (1999)). There are numerous TFs from plants and animals in the TALE superclass and the two main groups in plants are the KNOX and BEL types (Bürglin, “Analysis of TALE Superclass Homeobox Genes (MEIS, PBC, KNOX, Iroquois, TGIF) Reveals a Novel Domain Conserved Between Plants and Animals,” Nucleic Acids Res 25:4173-4180 (1997)). Related genes in animal systems play an important role in regulating gene expression.
Expression patterns and functional analysis of mutations support the involvement of knox genes in specific developmental processes of the shoot apical meristem. Kn1 from maize, the first plant homeobox gene to be discovered (Vollbrecht et al., “The Developmental Gene Knotted-1 is a Member of a Maize Homeobox Gene Family,” Nature 350:241-243 (1991)), is involved in maintenance of the shoot apical meristem and is implicated in the switch from indeterminate to determinate cell fates (Chan et al., “Homeoboxes in Plant Development,” Biochim Biophys Acta 1442:1-19 (1998); Kerstetter et al., “Loss-of-Function Mutations in the Maize Homeobox Gene, knotted1, are Defective in Shoot Meristem Maintenance,” Development 124:3045-3054 (1997); Clark et al., The CLAVATA and SHOOT MERISTEMLESS Loci Competitively Regulate Meristem Activity in Arabidopsis,” Development 122:1567-1575 (1996)). Transcripts of kn1 in maize (Jackson et al., “Expression of Maize KNOTTED1 Related Homeobox Genes in the Shoot Apical Meristem Predicts Patterns of Morphogenesis in the Vegetative Shoot,” Development 120:405-413 (1994)), OSH1 in rice (Sentoku et al., “Regional Expression of the Rice KN1-type Homeobox Gene Family During Embryo, Shoot, and Flower Development,” Plant Cell 11:1651-1663 (1999)), and NTH15 in tobacco (Tamaoki et al., “Ectopic Expression of a Tobacco Homeobox Gene, NTH15, Dramatically Alters Leaf Morphology and Hormone Levels in Transgenic Tobacco,” Plant Cell Physiol 38:917-927 (1997)) were localized by in situ hybridization to undifferentiated cells of the corpus and the developing stem, but were not detected in the tunica or leaf primordia. Overexpression of kn1 in Arabidopsis (Lincoln et al., “A knotted1-like Homeobox Gene in Arabidopsis is Expressed in the Vegetative Meristem and Dramatically Alters Leaf Morphology When Overexpressed in Transgenic Plants,” Plant Cell 6:1859-1876 (1994)) and in tobacco (Sinha et al., “Overexpression of the Maize Homeobox Gene, KNOTTED-1, Causes a Switch From Determinate to Indeterminate Cell Fates,” Genes Dev 7:787-795 (1993)), resulted in plants with altered leaf morphologies including lobed, wrinkled or curved leaves with shortened petioles and decreased elongation of veins. Plants were reduced in size and showed a loss of apical dominance. In plants with a severe phenotype, ectopic meristems formed near the veins of leaves indicating a reversion of cell fate back to the indeterminate state (Sinha et al., “Overexpression of the Maize Homeobox Gene, KNOTTED-1, Causes a Switch From Determinate to Indeterminate Cell Fates,” Genes Dev 7:787-795 (1993)). Overexpression of OSH1 or NTH15 in tobacco resulted in altered morphologies similar to the 35S-kn1 phenotype (Sato et al., “Abnormal Cell Divisions in Leaf Primordia Caused by the Expression of the Rice Homeobox Gene OSH1 Lead to Altered Morphology of Leaves in Transgenic Tobacco,” Mol Gen Genet. 251:13-22 (1996); Tamaoki et al., “Ectopic Expression of a Tobacco Homeobox Gene, NTH15, Dramatically Alters Leaf Morphology and Hormone Levels in Transgenic Tobacco,” Plant Cell Physiol 38:917-927 (1997)).
Alterations in leaf and flower morphology in 35S-NTH15 or OSH1 transgenic tobacco were accompanied by changes in hormone levels. Whereas levels of all the hormones measured were changed slightly, both gibberellin and cytokinin levels were dramatically altered (Kusaba et al., “Alteration of Hormone Levels in Transgenic Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1,” Plant Physiol 116:471-476 (1998); Tamaoki et al., “Ectopic Expression of a Tobacco Homeobox Gene, NTH15, Dramatically Alters Leaf Morphology and Hormone Levels in Transgenic Tobacco,” Plant Cell Physiol 38:917-927 (1997)). RNA blot analysis revealed that the accumulation of GA 20-oxidase1 mRNA was reduced several fold in transgenic plants (Kusaba et al., “Decreased GA1 Content Caused by the Overexpression of OSH1 is Accompanied by Suppression of GA 20-oxidase Gene Expression,” Plant Physiol 117:1179-1184 (1998); Tanaka-Ueguchi et al., “Overexpression of a Tobacco Homeobox Gene, NTH15, Decreases the Expression of a Gibberellin Biosynthetic Gene Encoding GA 20-oxidase,” Plant J 15:391-400 (1998)). A KNOX protein of tobacco binds to specific elements in regulatory regions of the GA 20-oxidase1 gene of tobacco to repress its activity (Sakamoto et al., KNOX Homeodomain Protein Directly Suppresses the Expression of a Gibberellin Biosynthesis Gene in the Tobacco Shoot Apical Meristern,” Genes Dev 15:581-590 (2001)). GA 20-oxidase is a key enzyme in the GA biosynthetic pathway necessary for the production of the physiologically inactive GA20 precursor of active GA1 (Hedden et al., “Gibberellin Biosynthesis: Enzymes, Genes and Their Regulation,” Annu Rev Plant Physiol Plant Mol Biol 48:431-460 (1997)). GA, and other active GA isoforms are important regulators of stem elongation, the orientation of cell division, the inhibition of tuberization, flowering time, and fruit development (Jackson et al., “Control of Tuberisation in Potato by Gibberellins and Phytochrome,” B. Physiol Plant 98:407-412 (1996); Hedden et al., “Gibberellin Biosynthesis: Enzymes, Genes and Their Regulation,” Annu Rev Plant Physiol Plant Mol Biol 48:431-460 (1997); Rebers et al., “Regulation of Gibberellin Biosynthesis Genes During Flower and Early Fruit Development of Tomato,” Plant J 17:241-250 (1999)).
Another plant homeobox gene family that is closely related to the knox genes is the BEL (BELL) family (Chan et al., “Homeoboxes in Plant Development,” Biochim Biophys Acta 1442:1-19 (1998); Bürglin, “Analysis of TALE Superclass Homeobox Genes (MEIS, PBC, KNOX, Iroquois, TGIF) Reveals a Novel Domain Conserved Between Plants and Animals,” Nucleic Acids Res 25:4173-4180 (1997)). BEL TFs have been implicated in flower and fruit development (Reiser et al., The BELL1 Gene Encodes a Homeodomain Protein Involved in Pattern Formation in the Arabidopsis Ovule Primordium,” Cell 83:735-742 (1995); Dong et al., “MDH1: an Apple Homeobox Gene Belonging to the BEL1 Family,” Plant Mol Biol 42:623-633 (2000)). Genetic analysis of BEL1 in Arabidopsis showed that expression of this TF regulated the development of ovule integuments and overlaps the expression of AGAMOUS (Ray et al., “Arabidopsis Floral Homeotic Gene BELL (BEL1) Controls Ovule Development Through Negative Regulation of AGAMOUS Gene (AG),” Proc Natl Acad Sci USA 91:5761-5765 (1994); Reiser et al., The BELL1 Gene Encodes a Homeodomain Protein Involved in Pattern Formation in the Arabidopsis Ovule Primordium,” Cell 83:735-742 (1995); Western et al., “BELL1 and AGAMOUS Genes Promote Ovule Identity in Arabidopsis thaliana,” Plant J 18:329-336 (1999)). In COP1 mutants, the photoinduced expression of ATH1, another BEL TF of Arabidopsis, was elevated, indicating a possible role in the signal transduction pathway downstream of COP1 (Quaedvlieg et al., “The Homeobox Gene ATH1 of Arabidopsis is Depressed in the Photomorphogenic Mutants cop1 and det1,” Plant Cell 7:117-129 (1995)).
Plants must maintain a great deal of flexibility during development to respond to environmental and developmental cues. Responses to these signals, which include day length, light quality or quantity, temperature, nutrient and hormone levels, are coordinated within the meristem (Kerstetter et al., “Shoot Meristem Formation in Vegatative Development,” Plant Cell 9:1001-1010 (1997)). In potato, there is a specialized vegetative meristem called the stolon meristem that develops as a horizontal stem and under inductive conditions will form the potato tuber (Jackson, “Multiple Signaling Pathways Control. Tuber Induction in Potato,” Plant Physiol. 119:1-8 (1999); Fernie et al., “Molecular and Biochemical Triggers of Potato Tuber Development,” Plant Physiol. 127:1459-1465 (2001)). Potato offers an excellent model system for examining how vegetative meristems respond to external and internal factors to control development at the molecular level. In model tuberization systems, synchronous tuber formation occurs under inductive conditions and shoot or stolon formation occurs under noninductive conditions. The cellular and biochemical processes that occur in these model systems have been examined extensively (Vreugdenhil et al., “Initial Anatomical Changes Associated with Tuber Formation on Single-Node Potato (Solanum tuberosum L.) Cuttings: A Re-evaluation,” Ann. Bot. 84:675-680 (1999); Xu et al., “The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation In vitro,” Plant Physiol. 117:575-584 (1998); Hannapel, “Characterization of Early Events of Potato Tuber Development,” Physiol. Plant 83:568-573 (1991); Wheeler et al., “Comparison of Axillary Bud Growth and Patatin Accumulation in Potato Leaf Cuttings as Assays for Tuber Induction,” Ann. Bot. 62:25-30 (1988)). In addition to being good systems to examine integration of signals at the meristem, understanding the molecular processes controlling tuberization in potato is important. Potato is the fourth largest crop produced in the world, ranking after maize, rice, and wheat, and is a major nutritional source in many countries (Jackson, “Multiple Signaling Pathways Control Tuber Induction in Potato,” Plant Physiol. 119:1-8 (1999); Fernie et al., “Molecular and Biochemical Triggers of Potato Tuber Development,” Plant Physiol. 127:1459-1465 (2001)); therefore, research focusing on the process of tuber initiation and development is very important.
Tuber formation in potatoes (Solanum tuberosum L.) is a complex developmental process that requires the interaction of environmental, biochemical, and genetic factors. Several important biological processes like carbon partitioning, signal transduction, and meristem determination are involved (Ewing et al., “Tuber Formation in Potato: Induction, Initiation and Growth,” Hort. Rev. 14:89-198 (1992)). Under conditions of a short-day photoperiod and cool temperature, a transmissible signal is activated that initiates cell division and expansion and a change in the orientation of cell growth in the subapical region of the stolon tip (Ewing et al., “Tuber Formation in Potato: Induction, Initiation and Growth,” Hort. Rev. 14:89-198 (1992); Xu et al., “Cell Division and Cell Enlargement During Potato Tuber Formation,” J. Expt. Bot. 49:573-582 (1998)). In this signal transduction pathway, perception of the appropriate environmental cues occurs in leaves and is mediated by phytochrome and gibberellins (van den Berg et al., “Morphology and (14C) gibberellin A-12 Metabolism in Wild-Type and Dwarf Solanum tuberosum ssp. Andigena Grown Under Long and Short Photoperiods,” J. Plant Physiol. 146:467-473 (1995); Jackson et al., “Phytochrome B Mediates the Photoperiodic Control of Tuber Formation in Potato,” Plant J. 9:159-166 (1996); Jackson et al., “Control of Tuberisation in Potato by Gibberellins and Phytochrome,” B. Physiol Plant 98:407-412 (1996)). Tuber development at the stolon tip is comprised of biochemical and morphological processes. Both are controlled by differential gene expression (Hannapel, “Characterization of Early Events of Potato Tuber Development,” Physiol. Plant 83:568-573 (1991); Bachem et al., “Analysis of Gene Expression During Potato Tuber Development,” Plant J. 9:745-753 (1996); Macleod et al., “Characterisation of Genes Isolated from a Potato Swelling Stolon cDNA Library,” Pot. Res. 42:31-42 (1999)) with most of the work focusing on the biochemical processes, including starch synthesis (Abel et al., “Cloning and Functional Analysis of a cDNA Encoding a Novel 139 kDa Starch Synthase from Potato (Solanum tuberosum L.),” Plant J. 10:981-991 (1996); Preiss, “ADPglucose Pyrophosphorylase: Basic Science and Applications in Biotechnology,” Biotech. Annu. Rev. 2:259-279 (1996); Geigenberger et al., “Overexpression of Pyrophosphatase Leads to Increased Sucrose Degradation and Starch Synthesis, Increased Activities of Enzymes for Sucrose-Starch Interconversions, and Increased Levels of Nucleotides in Growing Potato Tubers,” Planta 205:428-437 (1998)) and storage protein accumulation (Mignery et al., “Isolation and Sequence Analysis of cDNAs for the Major Potato Tuber Protein, Patatin,” Nucl. Acid Res. 12:7989-8000 (1984); Hendriks et al., “Patatin and Four serine Protease Inhibitor Genes are Differentially Expressed During Potato Tuber Development,” Plant Mol. Biol. 17:385-394 (1991); Suh et al., “Proteinase-Inhibitor Activity and Wound-Inducible Expression of the 22-kDa Potato-Tuber Proteins,” Planta 184:423-430 (1991)).
Much less is known about the morphological controls of tuberization, although it is clear that phytohormones play a prominent role (Koda et al., “Potato Tuber-Inducing Activities of Jasmonic Acid and Related Compounds,” Phytochemistry 30:1435-1438 (1991); Xu et al., “The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation In vitro,” Plant Physiol. 117:575-584 (1998), Sergeeva et al., “Tuber Morphology and Starch Accumulation are Independent Phenomena: Evidence from ipt-transgenic Potato Lines,” Physiol. Plant 108:435-443 (2000)). Gibberellins (GA), in particular, play an important role in regulating tuber development. High levels of GA are correlated with the inhibition of tuberization, whereas low levels are associated with the induction of tuber formation (Jackson et al., “Control of Tuberisation in Potato by Gibberellins and Phytochrome,” B. Physiol Plant 98:407-412 (1996); Xu et al., “The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation In vitro,” Plant Physiol. 117:575-584 (1998)). Specific genes, such as lipoxygenases (Kolomiets et al., “Lipoxygenase is Involved in the Control of Potato Tuber Development,” Plant Cell 13:613-626 (2001)) and MADS box genes (Kang et al., “Nucleotide Sequences of Novel Potato MADS-box cDNAs and their Expression in vegetative Organs,” Gene 166:329-330 (1995)) that are involved in regulating tuber formation have been identified.
Three independent research groups have recently confirmed that BEL-like TFs interact via protein binding with their respective knox-types in three separate species (Bellaoui et al., “The Arabidopsis BELL1 and KNOX TALE Homeodomain Proteins Interact Through a Domain Conserved Between Plants and Animals,” Plant Cell 13:2455-2470 (2001); Muiller et al., “In vitro Interactions Between Barley TALE Homeodomain Proteins Suggest a Role for Protein-Protein Associations in the Regulation of Knox Gene Function,” Plant J. 27:13-23 (2001); Smith et al., “Selective Interaction of Plant Homeodomain Proteins Mediates High DNA-Binding Affinity,” Proc. Nat'l. Acad. Sci. USA 99:9579-9584 (2002)), but to date, there is no published report on the function of this interaction. Moreover, nothing is known about the role of either KNOX or the BEL TFs in the regulation of development of tuberous plants, such as potato.
Plants adapt to their environment through the perception of external cues and the activation of signaling pathways. Two of the most important environmental cues to which plants respond are light quality and duration. Length of day or photoperiod is an example of an external cue that elicits developmental responses like germination, flowering, tuber formation, the onset of bud dormancy, leaf abscission, and cambial activity. Despite the significance of photoperiod in regulating growth responses, the precise signaling mechanism is unknown.
For communicating throughout the body of the organism, plants have evolved complex systems of signaling that may be transmitted in a volatile form or carried through the non-circulatory vascular system, the phloem and xylem. Signaling molecules include salts, sugars, carbohydrates, oxylipins, peptides, proteins, RNAs, and phytohormones. Little is known about this mechanism of long-distance transport in plants but exciting, innovative research is in progress (Lucas et al., “Selective Trafficking of KNOTTED1 Homeodomain Protein and its mRNA Through Plasmodesmata,” Science 270:1980-1983 (1995)). A model for intracellular RNA localization in the cell has been established for a number of animal and plant systems. Subcellular RNA movement in plants and animals is mediated by a complex transport system (Okita et al., “mRNA Localization in Plants: Targeting to the Cell's Cortical Region and Beyond,” Curr. Opin. Plant Biol. 5:553-559 (2002); Kloc et al, “Mechanisms of Subcellular mRNA Localization,” Cell 108:533-544 (2002)). Components of this system include a large ribonucleprotein (RNP) complex, facilitated movement of this RNP along microtubule or microfilament strands, and the anchoring of the RNA at its destination. Recognition and delivery of the RNA requires “zip code” elements and zip code proteins (Jansen, “mRNA Localization: Message on the Move,” Nat. Rev. Mol. Cell. Biol. 2:247-256 (2001)).
RNA transport in plants. Phloem sap contains a unique set of transcripts and proteins (Hoffmann-Benning et al., “Comparison of Peptides in the Phloem Sap of Flowering and Non-Flowering Perilla and Lupine Plants Using Microbore HPLC Followed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry,” Planta 216:140-147 (2002)). Analysis of pumpkin sap revealed the presence of an enriched, diverse population of RNAs. From a sap cDNA library, RNAs for transcription factors, cell cycle proteins, sucrose transporters, and proteins involved in intracellular vesicular trafficking and defense were identified (Ruiz-Medrano et al., “Phloem Long-Distance Transport of CmnNACPmRNA: Implications for Supracellular Regulation in Plants,” Development 126:4405-4419 (1999)). Three of these RNAs present in phloem sap moved selectively into apical tissues of heterografted scions. Two were transcription factors and one was a putative phloem transport protein. Experiments with phloem sap also showed that some ingredient in phloem exudate protected RNAs from degradation (Ruiz-Medrano et al., “Phloem Long-Distance Transport of CmNACPmRNA: Implications for Supracellular Regulation in Plants,” Development 126:4405-4419 (1999)). These studies clearly establish the presence of a system for the delivery of specific transcripts through the phloem to the shoot apical meristem.
There are numerous other examples of long-distance RNA movement in plants. Plant RNA viruses produce diverse proteins that facilitate cell-to-cell and long-distance movement by a variety of mechanisms (Gilbertson & Lucas, “How Do Viruses Traffic on the Vascular Highway?,” Trends Plant Science 1:260-268 (1996)). The RNA of sucrose transporter1 moves from companion cells through the plasmodesmata into the adjacent sieve elements (Kühn et al., “Macromolecular Trafficking Indicated by Localization and Turnover of Sucrose Transporters in Enucleate Sieve Elements,” Science 275:1298-1300 (1997)). There are several experimental examples demonstrating that cosuppression of expression mediated by systemic acquired gene silencing involves RNA transport within the phloem (Sonoda et al., “Grafi Transmission of Post-Transcriptional Gene Silencing: Target Specificity for RNA Degradation is Transmissible Between Silenced and Non-Silenced Plants, but not Between Silenced Plants,” Plant J. 21:1-8 (2000); Crete et al., “Graft Transmission of Induced and Spontaneous Post-Transcriptional Silencing of Chitinase Genes,” Plant J. 28:493-501 (2001)). This post-transcriptional epigenetic process is mediated by the sequence-specific degradation of targeted mRNAs (Meins, “RNA Degradation and Models for Post-Transcriptional Gene-Silencing,” Plant Mol. Biol. 43:261-273 (2000)).
Experiments involving heterografting showed that specific RNAs can move long distances through the phloem. Scions of cucumber grafted onto pumpkin stocks (lower portion of the graft) provided direct evidence that specific pumpkin mRNAs were translocated through the heterograft (Ruiz-Medrano et al., “Phloem Long-Distance Transport of CmNACPmRNA: Implications for Supracellular Regulation in Plants,” Development 126:4405-4419 (1999); Xoconostle-Cazares et al., “Plant Paralog to Viral Movement Protein that Potentiates Transport of mRNA into the Phloem,” Science 283:94-98 (1999)). The discovery of the RNA-binding protein, CmPP16, provided additional support for the long-distance transport of RNA in pumpkin (Xoconostle-Cazares et al., “Plant Paralog to Viral Movement Protein that Potentiates Transport of mRNA into the Phloem,” Science 283:94-98 (1999)). Microinjection and grafting studies demonstrated that CMPP16 moved from cell to cell, mediated the transport of RNA, and moved together with its mRNA into the sieve elements of scion tissue. One of the best examples of long-distance movement of RNA was reported for a KNOTTED-1-like homeobox gene of tomato, LeT6 (Kim et al., “Developmental Changes Due to Long-Distance Movement of a Homeobox Fusion Transcript in Tomato,” Science 293:287-289 (2001)). Heterografts were made with overexpression mutants exhibiting the characteristic Mouse-ear phenotype of KNOX gain-of-function mutants (Parnis et al., “The Dominant Developmental Mutants of Tomato, Mouse-ear and Curl, are Associated with Distinct Modes of Abnormal Transcriptional Regulation of a Knotted Gene,” Plant Cell 9:2143-2158 (1997)). The transport of this Knox RNA occurred in an acropetal direction and induced developmental changes in the wild-type scion consistent with the Mouse-ear phenotype. These results confirmed that the translocated RNA was functional. This mobile RNA accumulated in patterns specific to those observed for the native RNA, indicating that transport, and not promoter activity, may determine spatial expression. Remarkably, there are several examples of transcription factors, functional in meristems, with RNA that can be transported from cell to cell or over long distances (Kim et al., “Developmental Changes Due to Long-Distance Movement of a Homeobox Fusion Transcript in Tomato,” Science 293:287-289 (2001); Lucas et al., “Selective Trafficking of KNOTTED1 Homeodomain Protein and its mRNA Through Plasmodesmata,” Science 270:1980-1983 (1995); Ruiz-Medrano et al., “Phloem Long-Distance Transport of CmNACPmRNA: Implications for Supracellular Regulation in Plants,” Development 126:4405-4419 (1999); Haywood et al., “Plasmodesmata: Pathways for Protein and Ribonucleoprotein Signaling,” Plant Cell Supplement 303-325 (2002))
Mechanisms of transport. One important issue is to determine how RNAs can be recognized and delivered to specific sites in the plant body. Models established in animal systems may apply for phloem transport as well. Transported RNAs contain elements or structures in their RNA sequence that are recognized by RNA binding proteins (RBP). These recognition motifs are designated “zip codes.”
Zip codes can be short segments with a defined nucleotide sequence (Chan et al, “Fatvg Encodes a New Localized RNA that Uses a 25-Nucleotide Element (FVLE1) to Localize to the Vegetal Cortex of Xenopus Oocytes,” Development 126: 4943-4953 (1999)), repeated short signals, such as in the case of Vg1 or β-actin mRNA (Deshler et al., “Localization of Xenopus Vg1 mRNA by Vera Protein and the Endoplasmic Reticulum,” Science 276: 1128-1131 (1997); Kislauskis et al., “Sequences Responsible for Intracellular Localization of β-Actin Messenger RNA Also Affect Cell Phenotype,” J. Cell Biol. 127:441-451 (1994)), or stem-loop structures (Serano et al., “Small Predicted Stem-Loop Structure Mediates Oocyte Localization of Drosophila K10 Mrna,” Development 121:3809-3818 (1995); Chartrand et al., “Structural Elements Required For the Localization of ASH1 mRNA and of a Green Fluorescent Protein Reporter Particle In vivo,” Curr Biol 9: 333-336. (1999); Ramos et al., “RNA Recognition by a Staufen Double-Stranded RNA-Binding Domain,” EMBO J. 19:997-1009 (2000)). ASH1 mRNA of yeast is a stem-loop zip code element that lies in both the coding region (E1, E2) and in the 3′ UTR (E3) (Gonzalez et al., “ASH I mRNA Localization in Yeast Involves Multiple Secondary Structural Elements and Ashl Protein Myelin Basis Protein mRNA,” J. Cell Biol. 138:1077-1087 (1997)). ASH1 protein acts as a determinant to induce specific cell fates. Consequently, its localization in the cell is critical.
Localized mRNAs can contain more than one zip code that may have overlapping functions, or act in sequential targeting steps. Various maternal transcripts in Drosophila and Xenopus oocytes are localized through sequential events (Lasko, “RNA Sorting in Drosophila,” FASEB J. 13:421-433 (1999); Zhou et al., “Localization of Xcat-2 RNA, a Putative Germ Plasm Component, to the Mitochondrial Cloud in Xenopus Stage I Oocytes,” Development 122:2947-2953 (1996)). For example, the cell-fate determinant bicoid of Drosophila harbors a localization element in the 3′ UTR with a modular architecture. Bicoid mRNA undergoes several sequential transport steps, each involving different, partially overlapping regions in the highly structured 3′ UTR.
Zip Code Proteins. The cell interprets the information in a localization zip code via specific mRNA-binding proteins called zip code proteins. Although more than twenty-five zip codes have been characterized, zip-code-binding proteins are known for fewer than half. There are several examples of these types in animals: ZBP-1 (actin zip-code-binding protein) binds to the β-actin localization element in chicken fibroblasts. The Xenopus Vg1RBP (Vg1-mRNA-binding protein) is a homolog of ZBP-1 that recognizes the Vg1 mRNA zip code (Deshler et al., “A Highly Conserved RNA-Binding Protein for Cytoplasmic mRNA Localization in Vertebrates,” Curr. Biol. 8:489-496 (1998); Havin et al., “RNA-Binding Protein Conserved in Both Microtubule- and Microfilament-Based RNA Localization,” Genes Devel. 12:1593-1598 (1998)). She2 is a zip code protein that binds to the stem-loop-containing zip codes of yeast ASH1 mRNA (Bohl et al., “She2p, A Novel RNA-Binding Protein Tethers ASH1 mRNA to the Myo4p Myosin Motor via She3p,” EMBO J. 19:5514-5524 (2000); Long et al., “She2p is a Novel RNA-Binding Protein that Recruits the Myo4p/She3p Complex to ASH1 mRNA,” EMBO J. 19:6592-6601 (2000)). The Drosophila Staufen (Stau) protein is involved in localization of three transcripts (bcd, osk, and prospero) at three different stages of embryogenesis (Jansen, “mRNA Localization: Message on the Move,” Nat. Rev. Mol. Cell. Biol. 2:247-256 (2001)). Homologs of Drosophila Stau in other animal species, suggest a common mechanism for deciphering zip codes during mRNA localization. These protein/RNA interactions occur within the cell to regulate the delivery of key mRNAs for translation at specific sites.
Protein escorts. Whereas there is solid evidence that mRNAs are transported long distances in the plant, very little information is available on the mechanism of this transport. Relying on the animal model for subcellular movement of RNAs, it is becoming clear that RNA transport in plants is facilitated by escort or chaperone proteins. Numerous opportunities for protein interaction in transporting and targeting ribonucleoprotein complexes (RNPs) are clearly illustrated in a model described Lucas et al., “Selective Trafficking of KNOTTED1 Homeodomain Protein and its mRNA Through Plasmodesmata,” Science 270:1980-1983 (1995) (see Haywood et al., “Plasmodesmata: Pathways for Protein and Ribonucleoprotein Signaling,” Plant Cell Supplement 303-325 (2002)). The cell-to-cell transport of plant and viral RNPs involves delivery to the plasmodesmata (PD), modification of the PD microchannel, and partial unfolding of the protein or RNP complex. Movement through the PD could potentially involve chaperones, SEL (size exclusion limit)-recognition proteins, receptors, docking proteins, and transport proteins. There are numerous examples of proteins associated with cell-to-cell trafficking via the PD (Aoki et al., “A Subclass of Plant Heat Shock Cognate 70 Chaperones Carries a Motif that Facilitates Trafficking Through Plasmodesmata,” Proc. Natl. Acad. Sci. USA 99:16342-16347 (2002); Lee et al., “Selective Trafficking of Non-Cell-Autonomous Proteins Mediated by NtNCAPP1,” Science 299:392-396 (2003)). Transport proteins like sucrose transporter-1 and CmPP16 facilitate movement of RNAs from companion cells to sieve elements to deliver RNAs into the phloem (Kühn et al., “Macromolecular Trafficking Indicated by Localization and Turnover of Sucrose Transporters in Enucleate Sieve Elements,” Science 275:1298-1300 (1997); Xoconostle-Cazares et al., “Plant Paralog to Viral Movement Protein that Potentiates Transport of mRNA into the Phloem,” Science 283:94-98 (1999)). SUT1 mRNA actually moves through the phloem translocation stream (Haywood et al., “Plasmodesmata: Pathways for Protein and Ribonucleoprotein Signaling,” Plant Cell Supplement 303-325 (2002)). Putative zip code proteins, RNA-binding proteins in the phloem, may then deliver such signal RNAs to their site of activity in a specific organ. Viral movement proteins work in this way to enhance the transport of viral RNAs from cell to cell via the PD (Friedrich, “The Spread of Tobacco Mosaic Virus Infection: Insights into the Cellular Mechanism of RNA Transport,” Cell Mol. Life. Sci. 59:58-82 (2002); Fujiwara et al., “Cell-to-Cell Trafficking of Macromolecules Through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein,” Plant Cell 5:1783-1794 (1993); Lough et al., “Cell-to-Cell Movement of Potexviruses: Evidence for a Ribonucleoprotein Complex Involving the Coat Protein and First Triple Gene Block Protein,” Mol. Plant. Microbe Interact. 13:962-974 (2000)).
Fate of delivered RNA. Another mechanism for sorting mobile RNAs in plants as they reach terminal regions of the phloem is a recognition or surveillance field for screening movement into specific organs like the shoot or root apical meristem. It appears that plants control the exit of macromolecules from the phloem stream. Phloem-mobile endogenous RNA is trafficked selectively into the shoot apex. Support for this idea was obtained with studies that showed only specific phloem RNAs were detected in the apices of heterografted plants. RNAs that code for KNOX proteins of tomato moved across a graft in an acropetal direction to accumulate in the shoot apical meristems and leaf primordia of wild-type plants (Kim et al., “Developmental Changes Due to Long-Distance Movement of a Homeobox Fusion Transcript in Tomato,” Science 293:287-289 (2001)). Accumulation of these mRNAs was correlated with the phenotype of the gain-of-function mutant in wild-type scions. Further evidence is provided by the fact that viruses are prevented from invading cells of the apical meristem (Foster et al., “A Surveillance System Regulates Selective Entry of RNA into the Shoot Apex,” Plant Cell 14: 1497-1508 (2002)). Most viruses and long-distance post-transcriptional gene silencing (PTGS) signals are excluded from the shoot apex. These observations suggest the existence of an underlying filtering system. This surveillance system may regulate signaling and protect the shoot apex, in particular the cells that give rise to reproductive structures, from viral invasion. Despite data that support the existence of this surveillance system, very little is known about the molecular mechanisms that regulate this dynamic process. The most likely site for RNA surveillance is in the region between the protophloem and the meristem with a relay system through the PD (Lucas et al., “RNA as a Long-Distance Information Macromolecule in Plants,” Nat. Rev. Mol. Cell. Biol. 2:849-857 (2001)). The information relay may occur through a mobile ligand/membrane receptor system in conjunction with symplasmic movement through the cell.
The present invention is directed to overcoming these and other deficiencies in the art.